Expression and function of the UM4D4 antigen in human thymus

UM4D4 is a newly identified T cell surface molecule, distinct from the Ag receptor and CD2, which is expressed on 25% of peripheral blood T cells, resting or activated. Monoclonal anti-UM4D4 is mitogenic for T cells and T cell clones. Since alternative activation pathways independent of Ag/MHC recognition may be important in thymic differentiation, the expression and function of UM4D4 was examined in human thymus. UM4D4 was found on the surface of 6% of thymocytes. All thymocyte subsets contained UM4D4+ cells but expression was greatest on thymocytes that were CD1- (12%), CD3+ (11%) and especially CD4-CD8- (18%). CD3+CD4- CD8- cells, most of which bear the gamma delta-receptor, were greater than or equal to 50% + for UM4D4. Moreover, anti-UM4D4 was comitogenic for thymocytes together with PMA or IL-2. Anti-UM4D4 also reacted strongly with a subset of thymic epithelial cells in both cortex and medulla. Dual color fluorescence microscopy, with anti-UM4D4 and antibodies to other thymic epithelial Ag, showed UM4D4 expression on neuroendocrine thymic epithelium but not on thymic fibrous stroma. Thus, UM4D4 is expressed on, and represents an activation pathway for, a subset of thymic T cells. In addition, this determinant, initially identified as a novel T cell activating molecule, is broadly expressed by neuroendocrine thymic epithelium. Although the function of UM4D4 on the thymic epithelial cells is not yet clear, it is possible that UM4D4 represents a pathway for the functional activation of a subset of the thymic epithelium as well as a subset of thymocytes, thus playing a dual role in T cell differentiation.     

Sucrose synthase in rice plants : growth-associated changes in tissue specific distributions

Different parts of the rice (Oryza sativa L.) plant at different growth stages were analyzed for sucrose synthase (SS) by enzyme activity assay and enzyme-linked immunosorbent assay directly on the extracts or on the eluates from a gel filtration column. On a dry matter basis, the amount of soluble protein and SS activity decreased significantly, but the amount of enzyme protein changed little in growing leaves. In the grain, the SS activity was the highest at the early ripening stage and decreased later, but the amount of SS protein increased with the increase in maturity. In the root, a low activity of SS was detectable only in the tillering but not in other stages. Immunoblotting of SS protein extracted from different parts of rice showed two bands. Elution patterns of crude extracts from a gel filtration column showed the presence of several types of SS protein. Among them, two to three types with larger elution volumes had the SS activity but others with smaller elution volumes (considered as the aggregated forms) had no activity. The SS purified from different parts of the plant showed similar but distinctly different electrophoretic mobilities in a native gel. It has been concluded that different isozymes are expressed in different tissues at different growth stages.     

Modified, large-scale purification of the cytochrome o complex (bo-type oxidase) of Escherichia coli yields a two heme/one copper terminal oxidase with high specific activity.

The cytochrome o complex is a bo-type ubiquinol oxidase in the aerobic respiratory chain of Escherichia coli. This complex has a close structural and functional relationship with the eukaryotic and prokaryotic aa3-type cytochrome c oxidases. The specific activity, subunit composition, and metal content of the purified cytochrome o complex are not consistent for different preparative protocols reported in the literature. This paper presents a relatively simple preparation of the enzyme starting with a strain of Escherichia coli which overproduces the oxidase. The pure enzyme contains four subunits by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Partial amino acid sequence data confirm the identities of subunit I, II, and III from the SDS-PAGE analysis as the cyoB, cyoA, and cyoC gene products, respectively. A slight modification of the purification protocol yields an oxidase preparation that contains a possible fifth subunit which may be the cyoE gene product. The pure four-subunit enzyme contains 2 equivs of iron but only 1 equiv of copper. There is no electron paramagnetic resonance detectable copper in the purified enzyme. Hence, the equivalent of CuA of the aa3-type cytochrome c oxidases is absent in this quinol oxidase. There is also no zinc in the purified quinol oxidase. Finally, monoclonal antibodies are reported that interact with subunit II. One of these monoclonals inhibits the quinol oxidase activity of the detergent-solubilized, purified oxidase. Hence, although subunit II does not contain CuA and does not interact with cytochrome c, it still must have an important function in the bo-type ubiquinol oxidase.     

Preparation of lipid emulsions by pressure extrusion

Lipid emulsions consisting of a surface monolayer of phospholipid enclosing a core of neutral lipids have been prepared by repeated extrusion through polycarbonate filters of defined pore size. Particle size, as measured by photon correlation spectroscopy, decreases on successive passes through a 100 nm filter, reaching a near constant value (130-150 nm) after 4 passes. A corresponding decrease in the standard deviation of the particle size distribution occurs during this process. The recovery of lipids, especially of cholesterol and cholesterol ester, is improved if the emulsion is sonicated before extrusion through filters. [31P]-NMR and fluorescence techniques are used to confirm that the resulting structures are emulsions rather than lipid bilayers.     

Selective effect of zinc compared to other divalent metals on L-triiodothyronine binding to rat c-erbA alpha and beta proteins

The effects of zinc and other divalent metals on the [125I]T3 binding to rat c-erbA alpha and beta recombinant proteins were assessed. The addition of ZnCl2 caused a reversible and dose-dependent inhibition of [125I]T3 binding to rc-erbA beta proteins with half maximum inhibition occurring at 50-100 microM, but no significant effect on [125I]T3 binding to rc-erbA alpha under the same assay conditions. Scatchard analysis revealed a decrease in [125I]T3 binding capacity to beta protein without marked change in Kd values in presence of zinc. Moreover, significant inhibitions of [125I]T3 binding to both alpha and beta proteins were observed in the presence of 100 microM of either MnCl2, CdCl2 or CuCl2, but not MgCl2. Thus, the selective effect of zinc compared to other divalent metals to inhibit T3 binding to rc-erbA beta, but not alpha, proteins was documented and suggest a possible regulatory role for zinc in modulating the intracellular action of thyroid hormone.     

Papillary carcinoma of thyroid: classical and variants

Papillary carcinoma, the commonest primary cancer of thyroid, exhibits a broad morphological spectrum. In this review, the clinicopathological features of papillary carcinoma, classical and its variants (follicular, solid, cribriform, variant with exuberant nodular fasciitis-like stroma, encapsulated, diffuse sclerosing, diffuse follicular, tall cell, columnar cell, oxyphil cell, "dedifferentiated", occult, latent and microcarcinoma) are summarized.     

Eight small subunits of Euglena ribulose 1-5 bisphosphate carboxylase/oxygenase are translated from a large mRNA as a polyprotein.

The small subunit (SSU) of ribulose 1-5 bisphosphate carboxylase/oxygenase is a 15 kd protein in Euglena gracilis. The protein is synthesized as a 130 kd precursor as shown by immunoprecipitation of in vitro translation products and confirmed by immunoprecipitation of in vivo pulse-labeled Euglena proteins. From the published SSU amino acid sequence, an oligonucleotide was synthesized that specifically hybridizes to a large mRNA whose length (approximately 4.3 kb) is consistent with the precursor size. The complete nucleotide sequence of the SSU mRNA was obtained by sequencing a cDNA clone from a lambda gt11 library and completed by direct mRNA sequencing. We report for the first time the complete sequence of a large mRNA and show that it encodes eight consecutive SSU mature molecules. The deduced precursor amino acid sequence shows that the amino terminus of the first SSU molecule is preceded by a 134 amino acid peptide which is cleaved during the maturation process. This long transit peptide exhibits features characteristic of signal peptides involved in the secretion of proteins through the endoplasmic reticulum. This is in agreement with the idea that the third (outer) membrane of the Euglena chloroplast envelope is of endoplasmic reticulum origin.     

Distinct tyrosine phosphorylation sites in ZAP-70 mediate activation and negative regulation of antigen receptor function.

Biochemical and genetic evidence has implicated two families of protein tyrosine kinases (PTKs), the Src- and Syk-PTKs, in T- and B-cell antigen receptor signaling. ZAP-70 is a member of the Syk-PTKs that associates with the T-cell antigen receptor and undergoes tyrosine phosphorylation following receptor activation. Three tyrosine residues, Tyr-292, -492, and -493, have been identified as sites of phosphorylation following T-cell antigen receptor engagement. Utilizing ZAP-70- and Syk-deficient lymphocytes (Syk-DT40 cells), we provide biochemical and functional evidence that heterologous trans-phosphorylation of Tyr-493 by a Src-PTK is required for antigen receptor-mediated activation of both the calcium and ras pathways. In contrast, cells expressing mutations at Tyr-292 or -492 demonstrate hyperactive T- and B-cell antigen receptor phenotypes. Thus, phosphorylation of ZAP-70 mediates both activation and inactivation of antigen receptor signaling.     

Modulation of Calmodulin mRNA and Protein Levels in Barley Aleurone

Changes in calmodulin (CaM) mRNA and protein were investigated in aleurone layers of barley (Hordeum vulgare L. cv Himalaya) incubated in the presence and absence of calcium, gibberellic acid (GA3), and abscisic acid (ABA). CaM mRNA levels increased rapidly and transiently following incubation of aleurone layers in H2O, CaCl2, or GA3. The increase in CaM mRNA was prevented by ABA. This increase in CaM mRNA was brought about by physical stimulation during removal of the starchy endosperm from the aleurone layer. CaM protein levels did not increase in response to physical stimulation. Only incubation in GA3 plus CaCl2 brought about a rapid increase in CaM protein levels in the aleurone cell. ABA reduced the level of CaM protein below that found at the beginning of the incubation period. The rise in CaM protein preceded increases in the synthesis and secretion of [alpha]-amylase. Immunocytochemistry with monoclonal antibodies to carrot and mung bean CaM was used to localize CaM in aleurone protoplasts. Monoclonal antibodies to tubulin and polyclonal antibodies to tonoplast intrinsic protein and malate synthase were used as controls. CaM was localized to the nucleus, the vacuolar membrane, and the cytosol, but was not associated with microtubules.     

The LIM domain-containing Dbm1 GTPase-activating protein is required for normal cellular morphogenesis in Saccharomyces cerevisiae.

Normal cell growth in the yeast Saccharomyces cerevisiae involves the selection of genetically determined bud sites where most growth is localized. Previous studies have shown that BEM2, which encodes a GTPase-activating protein (GAP) that is specific for the Rho-type GTPase Rho1p in vitro, is required for proper bud site selection and bud emergence. We show here that DBM1, which encodes another putative Rho-type GAP with two tandemly arranged cysteine-rich LIM domains, also is needed for proper bud site selection, as haploid cells lacking Dbm1p bud predominantly in a bipolar, rather than the normal axial, manner. Furthermore, yeast cells lacking both Bem2p and Dbm1p are inviable. The nonaxial budding defect of dbm1 mutants can be rescued partially by overproduction of Bem3p and is exacerbated by its absence. Since Bem3p has previously been shown to function as a GAP for Cdc42p, and also less efficiently for Rho1p, our results suggest that Dbm1p, like Bem2p and Bem3p, may function in vivo as a GAP for Cdc42p and/or Rho1p. Both LIM domains of Dbm1p are essential for its normal function. Point mutations that alter single conserved cysteine residues within either LIM domain result in mutant forms of Dbm1p that can no longer function in bud site selection but instead are capable of rescuing the inviability of bem2 mutants at 35 degrees C.